Field of the Invention
The present invention relates to a method of expressing genes in yeast. More particularly, the invention relates to a method of expressing foreign genes in yeast. The invention further relates to composite DNA expression vectors capable of effecting ultrahigh expression of a polypeptide in a yeast host, yeast cells transformed by the DNA expression vector, use of the DNA expression vector to effect ultrahigh expression of a target polypeptide, and to target polypeptide products produced by yeast cells transformed by the DNA expression vectors.
Background of the Invention
The culture of various yeast strains has long been of great practical and economic importance. A description of commonly used yeasts and their characteristics may be found in Reminqton's Pharmaceutical Sciences, 16th Edition, A. Osol, ed., Mack Publishing Co., Easton, Penn., pp. 1276-1277 (1980). Large scale culture of this eukaryotic organism on an industrial scale is well known, Burrow, "Baker's yeast," p. 349-420, in, The Yeast, vol. 3, Rose and Harrison, eds., Academic Press, London (1970), and it has been suggested that existing know-how and technologies of large scale yeast culture might be advantageously adapted to the production of polypeptides. Loison et al., Bio/Technology 4:433-437 (1986). In addition, the use of yeasts such as Saccharomyces as hosts for expressing mammalian and other foreign proteins offers advantages lacking in more commonly used prokaryotic hosts such as Escherichia coli. For example, yeasts are capable of glycosylation while prokaryotic hosts such as E. coli are not. S. cerevisiae is generally regarded as safe by the United States Food and Drug Administration. The genetic system of S. cerevisiae has been well-defined. See, e.q., Mortimer et al., "Genetic Mapping in Saccharomyces cerevisiae," in, The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Strathern et al., eds., Cold Spring Harbor Laboratory, N.Y., pp. 11-26 (1981 ). The principles controlling gene expression in S. cerevisiae are also well characterized. See generally, Strathern et al., eds., The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, Cold Spring Harbor Laboratory, N.Y. (1982). Saccharomyces has thus recently been used to express such foreign polypeptides as hepatitis B, Valenzuela et al., Nature 298:347-350 (1982); Miyanohara et al., Proc. Natl. Acad. Sci. USA 80:1-5 (1983); Hitzeman et al., Nucleic Acids Res. 11:2745-2763 (1983); McAleer et al., Nature 307:178-180 (1984), interferons, Tuite et al., EMBO J. 1:603-608 (1982), calf chymosin, Mellor et al., Gene 24:1-14 (1983), epidermal growth factor, Urdea et al., Proc. Natl. Acad. Sci. USA 80:7461-7465 (1983), and calf prochymosin, Smith et al., Science 229:1219-1224 (1985).
In spite of the advantages of using a yeast such as Saccharomyces as an expression host, production of heterologous polypeptides generally has been disappointingly low as compared to that of the normal homologous gene products. Chen et al., Nucleic Acids Res. 12:8951-8970 (1984). Many investigators have sought to increase expression of proteins, especially of heterologous proteins, in yeast hosts by employing in an expression vector various components and combinations thereof that might be expected to increase heterologous protein expression because they are thought to strongly express homologous gene products in yeasts. Generally, these expression vectors have included a strong yeast promoter to direct transcription, a yeast sequence to terminate transcription, and a replication origin and selectable marker to maintain the plasmid in the host cell. The promoter and terminator sequences have generally been chosen from highly expressed yeast genes. Promoter sequences often have been selected based on their ability to tightly regulate expression of the foreign product which sometimes is toxic to the host cell.
For example, European Patent Application No. 84303833.2 (Pub. No. 0 128 743 A2) discloses a cloning vector containing a foreign gene and the yeast galactokinase (GAL1) regulatory region and promoter in position to regulate the expression of the foreign gene. This system was used to produce a galactokinase-bovine prochymosin fusion protein in S. cerevisiae. Also disclosed is the use of increased copy number for the GAL4 gene, known to enhance messenger RNA levels of the galactose metabolic genes in yeast, (Laughon et al., Proc. Natl. Acad. Sci. USA 79:6827-6831 (1982)), by isolating the GAL4 gene from yeast DNA and inserting it into a plasmid which contains the GAL1 regulatory region and promoter as well as the foreign gene.
European Patent Application No. 84101937.5 (Pub. No. 0 123 811 A2) discloses use of a GAL1 promoter of the yeast galactokinase gene in expressing a desired protein. The GAL1 promoter used was a DNA segment containing the transcription start signal for galactokinase in S. cerevisiae.
European Patent Application No. 84302723.6 (Pub. No. 0 123 544 A2) discloses use of the promoter and signal peptide portions of the yeast alpha-factor gene joined to the sequence encoding the desired heterologous protein. This system was used to express human interferon gamma, human serum albumin, bovine interferon alpha-1 and alpha-2, tissue plasminogen activator, rennin and a human insulin-like growth factor.
PCT Application No. WO 86/00926 discloses use of controllable repressor operator sequences responsive to the a 1/alpha-2 and alpha-2 gene products of the type a and type alpha yeast mating locus alleles to repress expression of a heterologous structural gene located downstream of the expression controlled sequence in the presence of a gene product of a yeast mating type locus.
PCT Application No. 83/01370 (Pub. No. WO 84/01153) discloses use of the SUC2 promoter, containing the transcription start signal for the structural gene for invertase to direct expression of an interferon gene within a yeast cell. The system allows passive regulation of interferon production, and was used to express human leukocyte interferon in S. cerevisiae.
European Patent Application No. 84104967.9 (Pub. No. 0 124 874 A2) discloses use of the 5'-flanking region from the yeast repressible acid phosphatase (PHO5) gene to tightly regulate the expression of heterologous genes in yeast. The PHO5 promoter/regulator was used to direct expression of interferon in S. cerevisiae.
European Patent Application No. 83110472.4 (Pub. No. 0 109 560 A2) discloses use of the promoter of the PHO5 gene, including the PHO5 signal peptide, to control expression of a target peptide gene in S. cerevisiae. The system was used to express norepinephrine alpha. Production of the target protein varied as a function of the phosphate concentration in the medium.
PCT Application No. 85/01470 (Pub. No. WO 86/00923) discloses use of a PHO5 promoter lying within the approximately 0.55 Kb sequence preceding the ATG translational start triplet at the beginning of the PHO5 coding sequence, which is excised and isolated from the PHO5 gene, to control expression of a heterologous peptide in S. cerevisiae. This PHO5 promoter system, in one embodiment containing also the major PHO5 start site, is used to direct expression of human fertility hormones, such as hCG, LH and FSH. Also disclosed is use of the promoter sequence of the gene encoding yeast glyceraldehyde-3'-phosphate alhydrogenase (GAPD) to control heterologous protein expression.
Hinnen et al., Chem. Ab. 102:Ab. No. 40931k (1985) discloses use of the promoter and 80% of the 5' end of the PHO5 signal sequence fused to a cDNA fragment encoding the mature protein sequence and a portion of the 3' end of the signal sequence of human alpha interferon. This system thus includes a hybrid PHO5-hIF-alpha signal sequence used to transform S. cerevisiae.
European Patent Application No. 83310471.6 (Pub. No. 0 109 559 A1) discloses use of the glyceraldehyde-3-phosphate dehydrogenase promoter to control expression of a target peptide in Saccharomyces. Also disclosed is the use of the 3'-terminal non-translational region of the GAPDH gene in the plasmid at a point downstream from the target peptide gene to increase production of the target peptide.
PCT Application No. 84/00153 (Pub. No. WO 84/04538) discloses use of a yeast regulon and a transcription terminator derived from one of the GAPDH genes of S. cerevisiae. It is disclosed that a 850 nucleotide GAPDH regulon was almost 100 times more effective as smaller regulons (280 nucleotides). Also disclosed is the use of the GAPDH regulon in conjunction with a signal sequence derived from a gene for yeast invertase, yeast acid phosphatase, unmatured forms of thaumatin-like proteins, unmatured forms of chymosin-like proteins or two consensus signal polypeptides, the amino acid sequences of which are disclosed. Also disclosed is the use of preferred yeast codons in either the target peptide sequence, the signal sequence or both.
Urdea et al., Proc. Natl. Acad. Sci. USA 80:7461-7465 (1983) discloses a plasmid containing the yeast GAPDH promoter, the structural gene for human epidermal growth factor (urogastrone) and a terminator derived from yeast alcohol dehydrogenase (ADH). This genetic unit was inserted into a yeast plasmid vector that contained the yeast 2 micron sequences, a DNA fragment containing the yeast LEU coding sequences, and a fragment of pBR322 containing the origin of replication and the ampicillin resistance gene, and used to direct synthesis of urogastrone. The DNA sequence for the gene encoding urogastrone was designed with yeast-preferred codons inferred from codon usage in the highly expressed yeast genes for GAPDH, ADH and pyruvate kinase. This expression system produced about 30 micrograms of target protein per liter of yeast culture.
European Patent Application No. 85870170.9 (Pub. No. 0 184 575 A2) discloses the use of strong yeast promoters such as the promoters of alcohol dehydrogenase (ADH1), enolase (EN08; ENO46), glyceraldehyde-3-phosphate dehydrogenase (GAP63; GAP491), phosphoglycerate kinase (PGK), alkaline phosphatase (PHO3; PHO5) and promoter p415, to control expression of enzymes of the 1,4-beta-N-acetylmuramidase type.
Bock et al., U.S. Pat. No. 4,517,294, disclose that a plasmid vector containing four components is required to express a heterologous gene such as the cDNA for human antithrombin III in yeast. The first component allows for transformation of both E. coli and yeast, and contains a selectable gene from each organism. The second component is a 5'-flanking sequence from a highly expressed yeast gene to promote transcription of a downstream-placed structural gene, such as the 5'-flanking sequence from yeast 3-phosphoglycerate kinase (PGK). The third component is a structural gene containing both an ATG translation start and translation stop signals. The fourth component is the 3'-flanking sequence of a yeast gene containing the signal for transcription termination and polyadenylation. Bock et al. further describe plasmids directing the production of antithrombin III in yeast.
In spite of these advances in the development of yeast vectors, expression of heterologous gene products has been much reduced compared with expression of homologous proteins in the same expression vector. Thus, Chen et al., Nucleic Acids Res. 12(23):8951-8970 (1984) noted that accounts of expression experiments involving interferons, hepatitis surface antigen, chymosin and human growth hormone reported heterologous protein levels far below the levels of normal homologous yeast gene products. This phenomenon, which is not well understood, often reduces the expression of the foreign gene product to milligrams or even micrograms per liter of cell culture. Such low production limits the utility of yeast as a host for heterologous protein expression. Chen et al. used the promoter sequence and transcription termination and polyadenylation sequence from the 3-phosphoglycerate kinase (PGK) gene to control expression of interferon in yeast, and reported expression 15-50 times lower than expression of the natural homologous PGK gene on the same plasmid. Chen et al. found that the difference was directly proportional to changes in steady-state levels of mRNAs, but could not explain the relationship.
Another suggested explanation for the relatively poor expression of heterologous genes in yeast has been that the codons used to direct protein synthesis in mammalian genes differ significantly from those used for highly expressed homologous yeast genes. It is well known that, because the genetic code is degenerate, several different codons can specify the inclusion of a given amino acid in a growing polypeptide chain. Nirenberg et al., "The RNA Code and Protein Synthesis," in, L. Frisch, ed., Symposia on Quantitative Biology XXXI, Cold Spring Harbor Laboratory, Publisher, N.Y. pp. 11-24 (1966). The highly expressed yeast genes contain a high proportion of specific codons which correspond to prominent tRNA species present in the cell. Genes which are expressed less efficiently generally include a more random codon choice for a particular amino acid. Bennetzen et al., J. Biol. Chem. 257:3026-3031 (1982). Since mammalian and other genes generally do not utilize these so-called "preferred" yeast codons, this might explain the observed limited expression. Urdea et al., supra, discussed above, used such an approach and reported enhanced expression of urogastrone under the control of a GAPDH promoter and ADH terminator in yeast. However, heterologous protein expression was low (about 30 micrograms/liter of culture) in comparison with expected homologous gene production.
In further refining yeast expression vectors to optimize production of foreign gene products, expression elements from different yeast genes have been combined to create hybrid expression vectors, such as those described hereinabove. Observed improvement in heterologous expression, however, generally has not been dramatic. For example, Smith et al., Science 229:1219-1224 (1985) employed many different combinations of promoter and secretion-signal sequences to control expression of prochymosin in yeast. Examination of total antigen production (see, Table 1 of Smith et al.) shows very little difference in prochymosin production among the various combinations employed, including a GAL1-PHO5 combination.
Thus, in spite of recent advances in yeast vector technology, a need has continued to exist for a DNA expression vector capable of effecting expression of heterologous polypeptides in yeast hosts at high expression levels.